Atherosclerosis is a principal cause of heart attack, stroke, hypertention and gangrene of the extremities and is (directly or indirectly) responsible for about 50% of all mortality in the United States, Western Europe and Japan (Ross, Nature 362:801-809, 1993). Atherosclerosis is a disease characterized by focal thickening of the inner portion of the artery wall, predisposing an individual to myocardial infarction (heart attack), cerebral infarction (stroke), hypertension (high blood pressure) and gangrene of the extremities. A common underlying events responsible for the formation of lesions are the intimal thickening of proliferating smooth muscle cells, probably in response to endothelial cell injury. Accumulation of smooth muscle cells in coronary arteries physically treated by angioplasty or by bypass surgery is also a prominent feature of restenosis. In addition to consisting primarily of proliferated smooth muscle cells, lesions of atherosclerosis are surrounded by large amounts of lipid-laden macrophages, varying numbers of lymphocytes and large amounts of connective tissue. PDGF is considered to be a principal growth-regulatory molecule responsible for smooth muscle cell proliferation. For instance, PDGF, as measured by mRNA analysis as well as in situ staining using an antibody against PDGF, was found within macrophages of all stages of lesion development in both human and nonhuman primate atherosclerosis (Ross et al., Science, 248: 1009-1012, 1990). PDGF was found in both non-foam cells and lipid rich macrophage foam cells. These data are consistent with PDGF playing a critical role in the atherosclerosis disease process. In addition, analysis of advanced human lesions examined by atherectomy catheter indicate that atherosclerotic and restenotic lesions contain high levels of PDGF as measured by in situ hybridization.
One of the principal surgical approaches to the treatment of atherosclerosis is transluminal angioplasty, or PCTA. Restenosis is due principally to the accumulation of neointimal smooth muscle cells, which is also a prominent feature of lesions of atherosclerosis. The failure rate due to restenosis after PCTA is 30-50% (Ross, Nature 362:801, 1993). Much of the restenosis is due to further inflammation, smooth muscle accumulation and thrombosis. Results from balloon angioplasty of a normal rat carotid artery, an animal model for PCTA, has demonstrated that PDGF and FGF may mediate intimal thickening that forms 7-21 days following injury (Ferns et al., Science 253:1129, 1991; Morisaki et al., Atherosclerosis 71:165, 1988; and Fingerle et al., Proc. Natl. Acad. Sci. USA 86:8412, 1989). Antibodies against bFGF prevented medial smooth muscle replication that occurred 24-48 hr. following injury of medial cells to the inflated balloon (Linder and Reidy, Proc. Natl. Acad. Sci. USA 88:3739, 1988). Antibodies against PDGF induced a statistically significant diminution in the neointimal accumulation of smooth muscle cells that resulted 3-6 days after ballooning (Ferns et al., Science 253:1129, 1991). Thus, similar growth factors are probably involved in both restenosis and atherogenesis. Inhibition of proliferative responses of these growth factors to smooth muscle cells can abrogate the diseases in animal models.
Restenosis is primarily due to proliferation of the neointimal smooth muscle cells rather than simple chemotaxis of smooth muscle cells. Pickering et al. (J. Clin. Invest. 91:1469-1480 1993) have shown in situ evidence of underlying proliferating smooth muscle cells in 11 of 11 restenotic lesions isolated from humans that had underwent angioplasty. The percentage of proliferating cells in some of the lesions approximated those found in human tumors and were observed up to 1 year following angioplasty. Further evidence that PDGF can play a role in arterial lesions was provided by Nabel et al. (J. Clin. Invest 91:1822-1829 1993) who showed that intimal thickening could be induced in porcine iliofemoral arteries by direct transfection and expression of a gene encoding PDGF. Thus, these data show that expression of PDGF within arteries in vivo can cause intimal hyperplasia and induce lesions relevant to pathogenesis of atherosclerosis and restenosis.
Occlusive lesions of atherosclerosis in humans can be reversed with aggressive treatment with lipid-lowering drugs (Brown et al., N. Engl. J. Med. 323:1289, 1990; and Kane et al., J. Am. Med. Assoc., 264:3007, 1990). Current therapies for inhibition of restenotic lesions are more limited, involving anticoagulants, steroids, fish oil with omega-3 fatty acids, anti-platelet therapies, and cytotoxic drugs, all with questionable benefit (Pompa et al., Circulation 84:1426, 1991). Other therapies have been tried in predictive in vivo models, including, for example, antibodies to FGF and PDGF, and conjugates of saporin and FGF (Casscells et al., Proc. Natl. Acad Sci. USA 89:7159-7163 1992).
Aberrant overexpression of FGF and PDGF has been found in synovial tissues from patients with rheumatoid arthritis. Overexpression was also found in various animal models of arthritis. These data implicate FGF and PDGF in inflammatory joint disease. RA is characterized by diffuse and nodular mononuclear cell infiltration and massive hyperplasia of the stromal connective tissues, comprised of fibroblast-like cells and new blood vessels. The highly invasive lesions resembles a localized nonmetastatic invasive neoplasm (Harris, Arthritis Rheum. 19:68-72, 1976). FGF directly stimulates angiogenesis in vivo. Both PDGF and FGF are mitogenic agents for synovial fibroblasts. Furthermore, PDGF can, in part, be produced by infiltrating monocytes, seen early in the pathogenesis of RA (Shimokado et al., Cell 43:277-286, 1985). In addition, PDGF can directly activate both neutrophils and monocytes, including release of superoxide, release of granule enzymes, and increased cell adherence and aggregation. These data suggest that PDGF is an important mediator of inflammatory responses in general (Tzeng et al., Blood, 66:179-183 1985).
Synovial tissues of RA patients express high levels of FGF and PDGF compared with synovial tissues of osteoarthritis patients, a non invasive joint disease (Sano et al., J. Cell. Biol. 110:1417-1426, 1990). These data are consistent with the theory that PDGF and FGF play a role in generating an invasive tumor-like behavior in arthritic joints of RA synovial connective tissues (Sano et al., J. Clin. Invest. 91:553-565 1993). Thus, there is a need in the art to develop an agent that can inhibit PDGF and FGF signaling of cellular activation and proliferation of synovial fibroblasts, activation of migration of PMN and monocytes. Such agents are useful agents for the treatment or prevention of progression of RA and other inflammatory-disease states involving monocytes, PMNs or platelets.
Neovascularization is critical for the growth for tumors and is important in a variety of angiogenic diseases, such as diabetic retinopathy, arthritis, psoriasis and haemangiomas. More than 70% of cancer patients die from metastatic dissemination of the initial tumor. Tumor neovascularization is the crucial process for survival of a primary tumor and for metastatic dissemination. Angiostatic steroids and heparin with anti-angiogenic agents such as protamin have been used as therapies to suppress tumor growth. These therapeutic approaches have serious limitations, because when the dose of heparin exceeded an optimum level for inhibition of angiogenesis, both tumor growth and angiogenesis were stimulated. Also, high doses of cortisone that are required for antiangiogenesis led to immunosuppression. Acquisition of an angiogenic phenotype marked a transition from hyperplasia to neoplasia (Folkman et al., Nature, 339:58-60 1989).
Both FGF and VEGF are potent angiogenic factors which induce formation of new capillary blood vessels. Transfection of human breast carcinoma cell line MCF-7 with FGF resulted in cell lines that form progressively growing and metastatic tumors when injected (s.c.) into nude mice. FGF may play a critical role in progression of breast tumors to an estrogen-independent, anti-estrogen resistant metastatic phenotype (McLeskey et al., Cancer Res. 53:2168-2177 1993). Breast tumor cells exhibited increased neovascularization, increased spontaneous metastasis and more rapid growth in vivo than did the non-transfected tumors. FGF has been shown to be transforming in NIH-3T3 cells and implicated in tumorigenesis and metastasis of mouse mammary tumors. FGF overexpression conferred a tumorigenic phenotype on a human adrenal carcinoma cell line suggesting that FGF's may also play a role in the transformation of epithelial cells. Polyclonal neutralizing antibodies to FGF inhibited tumor growth in Balb/c nude mice transplanted with K1000 cells (transfected with the leader sequence of bFGF) which form tumors in these mice (Hori et al., Cancer Res. 51:6180-9184 1991). Due to a role of FGF in neovascularization, tumorigenesis and metastasis there is a need in the art for FGF inhibitors as potent anti-cancer agents that exert their anti-FGF activity by preventing intracellular signaling of FGF.
VEGF is an endothelial cell-specific mitogen and an angiogenesis inducer that is released by a variety of tumor cells and expressed in human tumor cells in situ. Unlike FGF, transfection of cell lines with a cDNA sequence encoding VEGF, did not promote transformation, but did facilitate tumor growth in vivo (Ferrara et al., J Clin. Invest. 91:160-170 1993). This was likely due to paracrine stimulation of neovasculagenesis Furthermore, administration of polyclonal antibody which neutralize VEGF inhibited growth of human rhabdomyosarcoma, glioblastoma multiforme and leiomyosarcoma cell lines in nude mice (Kim et al., Nature 362:841-843 1993.)
Therefore, there is a need in the art to develop small molecule antagonists of PDGF, FGF, EGF and VEGF individually or as a group. Moreover, if these cytokines signal through a common second messenger pathway within the cell, such antagonists will have broad therapeutic activity to treat or prevent the progression of a broad array of diseases, such as coronary restenosis, tumor-associated angiogenesis, atherosclerosis, autoimmune diseases, acute inflammation, certain kidney diseases associated with proliferation of glomerular or mesangial cells, and occular diseases associated with retinal vessel proliferation. The present invention was made by discovering a common signaling mechanism, a group of active therapeutic agents, shown to be active by a large number of and variety of predictive assays, and discovering a common intracellular signaling intermediate.